This project investigates several aspects of DNA methylation and replication related to fragile X syndrome. The applicant and colleagues have observed that with repeat expansion the FMR1 gene is abnormally methylated (and thereby transcriptionally inactivated) and that its replication is abnormally delayed. The methylation pattern, as determined by protection from bisulfite conversion, showed a remarkable dichotomy: alleles are either highly methylated or not, with few intermediates. The degree of methylation varies, however, with some sites (apparently binding sites for transcription factors) less methylated than others. The methylation pattern is relatively conserved through cell division. DNA replication, as determined by BrdU incorporation in cells by sorted by DNA content and cyclin B1 staining, occurs quite late in the cell cycle, within 90 minutes of mitosis for FMR1 and as much as 1% of total genomic DNA. This observation cells into question the traditional concept of a gap (G2) between DNA replication and chromosome condensation for mitosis, and it may explain sites of chromosome fragility. In the renewal period, the applicants will further pursue the phenomena of DNA methylation and delayed replication. They will characterize the methylation pattern of the FMR1 region in different cell types at different stages of the cell cycle. They will examine clonal conservation of methylation patterns through cell division. They will study late DNA replication in cells sorted with an antibody to phosphorylated histone H3 and in conditions that promote chromosome fragility. Finally they will further develop and use a technique called "boomerang PCR" to determine the direction of DNA synthesis in relation to methylation.